Molecular Mechanisms for Gender Differences in Susceptibility to T Cell-Mediated Autoimmune in Nonobese Diabetic Mice This information is current as of September 30, 2021. Min Bao, Yang Yang, Hee-Sook Jun and Ji-Won Yoon J Immunol 2002; 168:5369-5375; ; doi: 10.4049/jimmunol.168.10.5369 http://www.jimmunol.org/content/168/10/5369 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Molecular Mechanisms for Gender Differences in Susceptibility to T Cell-Mediated Autoimmune Diabetes in Nonobese Diabetic Mice1

Min Bao,2 Yang Yang,2 Hee-Sook Jun, and Ji-Won Yoon3

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-␥, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early ؉ ␥ stages of insulitis at 4 wk of age. IFN- was significantly higher in young females, whereas IL-4 was higher in young males. CD4 Downloaded from T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-␥, but less IL-4, as compared with males. Treatment of CD4؉ T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-␥. We then examined whether the change in IL-12-induced IFN-␥ production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the

development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of http://www.jimmunol.org/ IL-12-induced STAT4 activation by estrogen. The Journal of Immunology, 2002, 168: 5369Ð5375.

ype 1 diabetes, also known as insulin-dependent diabetes mice. The incidence of diabetes was significantly increased in male mellitus, results from the loss of insulin-producing pan- NOD mice, but decreased in females, by castration at the time of creatic ␤ cells by ␤ cell-specific, cell-mediated autoim- weaning (12). The basal circulating levels of estrogen were found T 4 mune responses (1Ð3). Nonobese diabetic (NOD) mice spontane- to be about twice as high in female NOD mice as in other strains ously develop autoimmune and are considered to of mice (13). In addition, the long-term administration of androgen be one of the best animal models for human insulin-dependent or its derivatives to young female NOD mice resulted in a decrease diabetes mellitus (4). The destruction of pancreatic ␤ cells in NOD in the incidence of diabetes (14Ð16). However, it is poorly under- by guest on September 30, 2021 mice is preceded by infiltration of dendritic cells/macrophages and stood how sex hormones modulate the incidence of diabetes in then T and B cells into the pancreatic islets (5Ð10). Infiltration of NOD mice. This investigation was initiated to find mechanisms by immunocytes into the periislet region of the pancreas (periinsulitis) which sex hormones affect the development of autoimmune dia- begins at 3Ð4 wk of age, followed by the slow, progressive, and betes in NOD mice. We found that estrogen increased the IL-12- selective destruction of insulin-producing ␤ cells at 4Ð6 mo of age induced activation of STAT4, which enhanced the Th1 immune (11). Both female and male NOD mice show insulitis; however, response, whereas testosterone (TS) did not significantly change females exhibit more invasive and destructive insulitis, leading to the activation of STAT4. Thus, sex hormones may modulate the an earlier onset (12 wk of age) and higher incidence (80Ð90%) of Th1/Th2 immune balance through the regulation of IL-12-induced diabetes as compared with males (20 wk of age, 10Ð30%). STAT4 activation in the early stages of the T cell-mediated auto- It has been suggested that sex hormones are associated with the immune process and affect the development of diabetes in sexual dimorphism in the onset of autoimmune diabetes in NOD NOD mice. Materials and Methods Department of Microbiology and Infectious Diseases and Laboratory of Viral and Mice Immunopathogenesis of Diabetes, Julia McFarlane Diabetes Research Center, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada All mice used in this study were purchased from Taconic Farms (German- Received for publication January 15, 2002. Accepted for publication March 15, 2002. town, NY) and maintained in the pathogen-free facility in the Health Sci- ence Center of the University of Calgary (Calgary, Alberta, Canada). The The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance use and care of the animals in this study were approved by the Animal Care with 18 U.S.C. Section 1734 solely to indicate this fact. Committee, Faculty of Medicine, University of Calgary. 1 This work was supported by a grant from the Canadian Institutes of Health Research Isolation of islets (MA 9584). Y.Y. is a recipient of the Career Development Award from the Juvenile Diabetes Research Foundation International. J.-W.Y. holds a Canada Research Chair Islets were isolated from female and male NOD mice at 4, 8, 16, and 20 wk in Diabetes. of age and from C57BL/6 mice at 20 wk of age by collagenase digestion 2 M.B. and Y.Y. contributed equally to this work. and Ficoll gradient centrifugation (Sigma-Aldrich, St. Louis, MO), as de- scribed elsewhere (17), and pooled for RNA extraction. Each group con- 3 Address correspondence and reprint requests to Dr. Ji-Won Yoon, Laboratory of Viral and Immunopathogenesis of Diabetes, Julia McFarlane Diabetes Research Cen- tains pooled islet RNA from four to six mice. ter, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Isolation of CD4ϩ T cells Alberta, Canada T2N 4N1. E-mail address: [email protected] 4 Abbreviations used in this paper: NOD, nonobese diabetic; E2, 17␤-estradiol; ER, Lymphocytes were prepared from the lymph nodes of female and male ϩ estrogen receptor; TS, testosterone; TSR, TS receptor. NOD mice at 4 and 10 wk of age, and CD4 T cells were purified using

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 5370 ROLE OF SEX HORMONES IN AUTOIMMUNE DIABETES the MACS system anti-CD4 beads (Miltenyi Biotec, Mu¬nchen, Germany). ice for 20 min and centrifuged at 12,000 ϫ g for 20 min at 4¡C. The cleared The purity was Ͼ90% when determined by flow cytometric analysis. lysates were then immunoprecipitated with anti-STAT4 or anti-STAT6 Ab (Upstate Biotechnology, Lake Placid, NY). The precipitated protein was RT-PCR analysis of gene expression resolved on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were first probed Total RNA was extracted from islets or cultured lymphocytes using TRIzol with anti-phosphotyrosine Ab (4G10; Upstate Biotechnology) and visual- reagent (Life Technologies, Burlington, Ontario, Canada), according to the ized by the ECL system (Amersham Life Science, Arlington Heights, IL). manufacturer’s protocol. A total of 3 ␮g RNA was subjected to the first- The membranes were subsequently stripped using a solution containing strand cDNA synthesis in 20 ␮l reaction mixture containing 50 mM Tris- 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 8), and reprobed with HCl (pH 8.3), 75 mM KCl, 3 mM MgCl ,10␮g/ml oligo(dT), 10 mM 2 anti-STAT4 or anti-STAT6 Ab (Upstate Biotechnology). DTT, 0.5 mM of each nucleotide, 2 U/ml RNase inhibitor, and 2.5 U Superscript II reverse transcriptase (Life Technologies) at 37¡C for 60 min. Statistical analysis The samples were then heated at 95¡C for 5 min to inactivate the enzymes and diluted to 100 ␮l with distilled water. Five microliters of serially di- Student’s t test was used to calculate statistical significance in all experi- luted cDNA samples were added to 15 ␮l reaction mixture containing 0.2 ments. A value of p Ͻ 0.05 was considered to be significant. U Taq DNA polymerase (Sigma-Aldrich) for PCR. The reaction conditions were optimized for each pair of primers. Primers were as follows: ␤-actin Results sense (5Ј-GTTACCAACTGGGACGACA-3Ј) and antisense (5Ј-TGGC CATCTCCTGCTCGAA-3Ј); IFN-␥ sense (5Ј-AGCTCTGAGACAAT Preferential expression of Th1 cytokines in islet infiltrates of GAACGC-3Ј) and antisense (5Ј-GGACAATCTCTTCCCCACCC-3Ј); young female NOD mice Ј Ј Ј IL-4 sense (5 -TCTTTCTCGAATGTACCAGG-3 ) and antisense (5 - To determine whether there is a difference in the level of expres- CATGGTGGCTCAGTACTACG-3Ј); estrogen receptor (ER)-␣ sense (5Ј- Ј Ј sion of Th1 (IFN-␥) and Th2 (IL-4) cytokines in islet-infiltrating GAGACTGTCCAGCAGTAACGAGAA-3 ) and antisense (5 -GGACAA Downloaded from GGCAGGGCTATTC-3Ј); and TS receptor (TSR) sense (5Ј-TCTCAAGA lymphocytes between female and male NOD mice, we isolated GTTTGAATGGCTCC-3Ј) and antisense (5Ј-GAGATGATCTCTGCC islets from female or male NOD mice at 4, 8, 16, and 20 wk of age Ј ATCATTTC-3 ). The PCR products were visualized on a 1.5% agarose gel and examined the expression of IFN-␥ and IL-4 by RT-PCR anal- by ethidium bromide staining, and the densitometric analysis of the PCR ␥ products was performed by Adobe Photoshop 4.1 software (Adobe Sys- ysis. The expression of IFN- and IL-4 was readily detected in the tems, Mountain View, CA). islets from all ages tested of NOD mice, whereas the expression of these cytokines was not detected in the islets from C57BL/6 mice T cell proliferation assay (Fig. 1A). The expression of IFN-␥ increased with increasing age http://www.jimmunol.org/ The isolated CD4ϩ T cells from lymph nodes were washed and resus- in both female and male NOD mice up to 16 wk of age, with pended in serum-free RPMI 1640 medium. The cells (5 ϫ 105 cells/well) females showing significantly higher expression of IFN-␥ than were seeded into 96-well round-bottom culture plates coated with 0, 1.25, 2.5, or 5 ␮g/ml anti-CD3 Ab (145.2C11; BD PharMingen, San Diego, CA) and incubated for 72 h in the presence of 2 ␮g/ml anti-CD28 Ab (BD PharMingen). [3H]Thymidine (1 ␮Ci) was added to each well, and the cells were cultured for an additional 16 h. The cells were harvested to determine the incorporation of [3H]thymidine.

ELISA for cytokine production by guest on September 30, 2021 CD4ϩ T cells were activated with anti-CD3 and anti-CD28 Ab for 72 h, as described above. The culture supernatant was collected, and the production of IFN-␥ and IL-4 was determined by ELISA using Duoset ELISA devel- opment system (R&D Systems, Minneapolis, MN), according to the man- ufacturer’s protocol. IL-4- and IL-12-induced activation of CD4ϩ T cells CD4ϩ T cells (2.5 ϫ 106/ml) isolated from lymph nodes of 4- and 10-wk- old female or male NOD mice were activated with Con A (2.5 ␮g/ml; Sigma-Aldrich) and IL-2 (20 U/ml; Takeda Chemical Industries, Osaka, Japan) for 3 days. The cells were then washed and reincubated in serum- free RPMI medium in the presence of IL-2 for another 4 days. The cells were washed again and incubated in serum-free culture medium with or without 17␤-estradiol (E2, 25 ng/ml; Sigma-Aldrich) or TS (25 ng/ml) for 20 h. IL-12 (20 ng/ml; PeproTech, Rocky Hill, NJ) was added to the culture medium, and the cells were incubated for an additional 48 h. The super- natant was collected for cytokine ELISA, and cells were harvested for RNA extraction to perform RT-PCR analysis. For immunoprecipitation and immunoblot analysis of STAT4 activation, the cells (5 ϫ 106/ml) were incubated for 20 min, instead of 48 h, in the presence of IL-12 (20 ng/ml). For analysis of STAT6 activation, cells were incubated with IL-4 (40 ng/ ml), instead of IL-12, for 40 min after sex hormone treatment. Flow cytometric analysis of IL-12R expression in CD4ϩ T cells CD4ϩ T cells were activated with Con A, rested, and treated with E2 or TS, as described above. The cells were harvested and incubated with purified ␥ anti-mouse IL-12R ␤1 Ab (BD PharMingen). The cells were washed and FIGURE 1. RT-PCR analysis of IFN- and IL-4 gene expression in islet incubated with biotinylated anti-mouse IgG, followed by incubation with infiltrates from female and male NOD mice. Islets were isolated from fe- streptavidin-PerCP. The expression of IL-12R was measured by flow male and male NOD mice and C57BL/6 (B6) mice at the indicated ages, cytometry. and the expression of IFN-␥ and IL-4 was examined by RT-PCR (A) and normalized against the expression of ␤-actin, an internal standard (B and Immunoprecipitation and immunoblotting C). IFN-␥/IL-4 is calculated as a ratio of IFN-␥ PCR product to IL-4 PCR The cells were washed and lysed in ice-cold lysis buffer containing 1% product amplified from the same cDNA (D). Representative gel pictures Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 30 (A) or mean Ϯ SD (BÐD) of three independent experiments are shown. .p Ͻ 0.05, comparing females to males ,ء mM aprotinin, and 100 mM sodium orthovanadate. The lysates were left on The Journal of Immunology 5371 males at 4 wk of age (Fig. 1, A and B). The expression of IL-4 pared with 4 wk of age (Fig. 2, CÐF). There were considerable increased between 4 and 8 wk of age and plateaued thereafter in differences in the production of IFN-␥ and IL-4 between T cells both males and females. At all ages tested, the expression of IL-4 from female and male NOD mice. At 4 wk of age, CD4ϩ T cells was significantly higher in males than in females (Fig. 1, A and C). from female NOD mice produced significantly higher amounts of When we examined the ratio of IFN-␥ and IL-4 gene expression, IFN-␥ (Fig. 2C), but lower amounts of IL-4 (Fig. 2E), compared it was found to be significantly higher in females than in males at with CD4ϩ T cells from males. This difference was more pro- 4 and 8 wk of age (Fig. 1D). nounced when CD4ϩ T cells were activated with higher concen- ␥ ϩ trations of anti-CD3 Ab. At 10 wk of age, the amount of IFN- Differential Th1/Th2 cytokine production in CD4 T cells of produced by CD4ϩ T cells from female NOD mice was still higher young female and male NOD mice than that from males (Fig. 2D), but the production of IL-4 was To determine whether there is a difference in Th1/Th2 cytokine almost equal between females and males (Fig. 2F). When we cal- production in T cells of female and male NOD mice, we isolated culated these results as a ratio of IFN-␥:IL-4, we found that this CD4ϩ T cells from lymph nodes of young (4-wk-old) and adult ratio was significantly higher in female mice than in male mice at (10-wk-old) NOD mice of both sexes and activated them with 4 wk of age (Fig. 2G), whereas it was not different between fe- anti-CD3 and anti-CD28 Abs in serum-free medium, to avoid any males and males at 10 wk of age (Fig. 2H). These results suggest contamination by sex hormones. We then examined the prolifer- that the gender-associated activation of the Th1 response, as indi- ative response of the cells and the production of IFN-␥ and IL-4. cated by the up-regulation of IFN-␥, occurs predominantly in the We found that CD4ϩ T cells from both female and male mice at T cells of young mice as compared with adult mice.

ϩ Downloaded from both ages proliferated equally well at all concentrations of anti- The expression of IFN-␥ and IL-4 in CD4 T cells was also CD3 Ab (Fig. 2, A and B), but T cells from both female and male examined at the mRNA level by semiquantitative RT-PCR. The NOD mice produced more IFN-␥ and IL-4 at 10 wk of age com- expression of IFN-␥ was higher in females than in males at both 4 and 10 wk of age. However, the expression of IL-4 was higher in males than in females at 4 wk of age (Fig. 3, A and C). The ratio of IFN-␥:IL-4 mRNA was significantly higher in females than in

males at 4 wk of age (Fig. 3B), but was not different at 10 wk of http://www.jimmunol.org/ age (Fig. 3D), consistent with the production of these cytokines at the protein level. This result indicates that gender factors influence the expression of IFN-␥ and IL-4 at the transcriptional level. To determine whether this differential expression in T cells between female and male mice is due to different levels of expression of sex hormone receptors, we examined the expression of ER and TSR in CD4ϩ T cells from 4- and 10-wk-old NOD mice. We found no difference in the expression level of sex hormone receptors in the T cells between males and females at any age (Fig. 4). by guest on September 30, 2021

Increase of IL-12-induced production of IFN-␥ in T cells by estrogen treatment and decrease by TS treatment As we found that there was a gender difference in the production of IFN-␥ at 4 wk of age, but not at 10 wk of age, we asked whether exposure to TS or E2 in vitro might reveal a gender difference in cytokine production by CD4ϩ T cells from 10-wk-old NOD mice. Purified CD4ϩ T cells from lymph nodes from 10-wk-old female or male NOD mice were activated with Con A or anti-CD3 Ab in the presence of E2 or TS. However, we found no difference in the production of IFN-␥ and IL-4 between T cells from female or male mice treated with or without E2 or TS (data not shown). We then asked whether prolonged cell proliferation and rest in the absence of sex steroids would result in a response to subsequent sex steroid treatment of CD4ϩ T cells from 10-wk-old mice. In this experi- ment, CD4ϩ T cells from 10-wk-old female NOD mice were first activated with Con A for 3 days, rested in IL-2-containing serum- free medium for another 4 days, and exposed to E2 or TS for 20 h before being stimulated with IL-12, a potent inducer of Th1 cyto- FIGURE 2. Production of IFN-␥ and IL-4 in CD4ϩ T cells of female kines. We found that steroid treatment did not affect the prolifer- and male NOD mice. CD4ϩ T cells from lymph nodes of 4-wk-old (A, C, ative response of the T cells to Con A and IL-12 in male or female E, and G) and 10-wk-old (B, D, F, and H) mice were activated with the NOD mice (data not shown). However, we found that E2-treated T indicated concentrations of immobilized anti-CD3 Ab and 2 ␮g/ml anti- cells from both males and females clearly showed an increased 3 CD28 Ab for 72 h. Proliferation was measured by [ H]thymidine incorpo- production of IFN-␥, whereas TS-treated T cells from females, but ration (A and B). The production of IFN-␥ (C and D) and IL-4 (E and F) ␥ ␥ not males, showed a decreased production of IFN- compared with was determined by ELISA. The ratio of IFN- and IL-4 production (G and ␥ H) is expressed as a ratio of the amount of IFN-␥ to the amount of IL-4 untreated IL-12-stimulated T cells (Fig. 5A). IFN- mRNA ex- produced from the same T cells. Representative results are shown as the pression was also increased by estrogen treatment and decreased mean Ϯ SD for triplicate samples. Similar results were obtained for all by TS treatment in female NOD mice (Fig. 5, B and C). Similar ϩ p Ͻ 0.005, comparing results were obtained for CD4 T cells from both female and male ,ءء ;p Ͻ 0.01 ,ء .three independent experiments females with males for each treatment. NOD mice; therefore, only the data from female mice are shown 5372 ROLE OF SEX HORMONES IN AUTOIMMUNE DIABETES

FIGURE 3. The expression of IFN-␥ and IL-4 mRNA in CD4ϩ T cells of female and male NOD mice. CD4ϩ T cells isolated from 4-wk-old (A and B) and 10-wk-old (C and D) female and male NOD mice were activated with the indicated concentrations of anti- CD3 Ab and 2 ␮g/ml anti-CD28 Ab. IFN-␥ and IL-4 gene expression was analyzed by RT-PCR (A and C) and normalized against ␤-actin expression (B and D). The IFN-␥ and IL-4 gene expression is expressed as ratio of IFN-␥ PCR product to IL-4 product amplified from the same cDNA. Representative gel pictures (A and C) or mean Ϯ SD (B and D) of three independent -p Ͻ 0.01, comparing fe ,ء .experiments are shown males with males for each treatment. Downloaded from

in Fig. 5, B and C. These results indicate that sex hormones di- compared with untreated T cells (Fig. 7, A and B). When we rectly regulate IL-12-induced cytokine production of T cells. examined the effect of sex hormones on IL-4-induced activation To determine whether the IL-12-induced change in IFN-␥ pro- of STAT6, which play a pivotal role in Th2 differentiation (20, duction in E2- or TS-treated T cells was due to a change in IL-12R 21), we found no change in the phosphorylation of STAT6 in http://www.jimmunol.org/ expression, which is up-regulated in Th1 cells, we examined the IL-4-stimulated T cells in the presence of estrogen or TS (Fig. gene expression of IL-12R ␤1 in T cells activated by Con A and 7, C and D). IL-2 and treated with E2 or TS by flow cytometric analysis. We found that treatment with E2 or TS did not significantly change the expression of the IL-12R (Fig. 6A). In addition, we examined the Discussion expression of sex hormone receptors by RT-PCR and found that It has been known that gender influences the susceptibility to var- treatment with sex hormones did not affect the expression of E2 or ious autoimmune diseases (22). Females are generally more sus- TS receptors in these cells (Fig. 6B). ceptible to diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Sjo¬gren’s syndrome, myasthenia by guest on September 30, 2021 Increase of IL-12-induced STAT4 activation in T cells by gravis, and experimental allergic encephalomyelitis. Although the estrogen treatment incidence of type 1 diabetes is similar in both men and women, the To determine whether the differential expression of IFN-␥ in IL- NOD mouse, an animal model of type 1 diabetes, shows female 12-stimulated T cells results from the modulation of the activation prevalence of the disease. Castration of male NOD mice increases, of STAT4, which is known to play a key role in IL-12-induced whereas castration of female NOD mice decreases the frequency IFN-␥ expression (18, 19), CD4ϩ T cells were activated with Con of autoimmune diabetes, and treatment of female NOD mice with A, rested, and then incubated with E2 or TS for 20 h. After incu- TS prevented the development of diabetes (14Ð16). However, the bation of these cells with IL-12 for 20 min, we examined the level mechanisms for the gender differences in susceptibility to autoim- of phosphorylated STAT4 by Western blot. We found that T cells mune diabetes are poorly understood. treated with E2 had increased phosphorylation of STAT4, whereas The immune systems of females and males have different char- this was not significantly different in T cells treated with TS as acteristics. For example, higher Ab production after immunization was observed in female mice, women have higher absolute num- bers of CD4ϩ T lymphocytes (23), and higher Th1 cytokine pro- duction was observed in female mice (24). In addition, sex hor- mones were shown to modulate the cytokine profiles in immunocytes (25) and T cell lines and clones (26, 27) in vitro. As well, the expression of cytokine genes was found to be influenced by sex hormones in an animal model of experimental autoimmune encephalitis. The production of Th1 cytokines such as IL-12 and IFN-␥ was significantly less in lymph node cells from male as compared with female mice after Ag-specific stimulation (28). Therefore, it is conceivable that sex hormones may contribute to

ϩ the gender difference in the development of diabetes in NOD mice FIGURE 4. Expression of E2 and TSR genes in CD4 T cells from by influencing the balance of Th1/Th2 cytokine production, as it is female and male NOD mice. A, ER-␣ and TSR gene expression was de- known that the Th1 response plays a pathogenic role and the Th2 tected by RT-PCR from T cells of 4-wk-old female (lane 1), 4-wk-old male (lane 2), 10-wk-old female (lane 3), and 10-wk-old male (lane 4) NOD response plays a preventive role in the development of autoim- mice. ER-␣ gene expression (B) or TSR gene expression (C) is expressed mune diabetes in NOD mice (29Ð33). as a ratio of PCR product to ␤-actin product amplified from the same We first examined the expression of IFN-␥, as a characteristic cDNA. Representative gel pictures (A) or mean Ϯ SD (B and C) of three Th1 cytokine, and IL-4, as a characteristic Th2 cytokine, in the independent experiments are shown. islet infiltrates in female and male NOD mice at various ages. We The Journal of Immunology 5373 Downloaded from http://www.jimmunol.org/

FIGURE 6. Expression of IL-12R and ER and TSR genes in E2- or ϩ ϩ FIGURE 5. IL-12-induced IFN-␥ production in CD4ϩ T cells of NOD TS-treated CD4 T cells. CD4 T cells of 10-wk-old female NOD mice mice treated with E2 or TS. CD4ϩ T cells (2.5 ϫ 106 cells/ml) were were activated with Con A and rested in serum-free medium, then treated activated with Con A and rested in serum-free medium, followed by in- with E2 (25 ng/ml) or TS (25 ng/ml), as described in Materials and Meth- ␤ cubation in E2 (25 ng/ml) or TS (25 ng/ml) and activation with IL-12 (20 ods. A, Expression of IL-12R 1 was measured by flow cytometry. B, ␣ ng/ml) for 48 h. As controls, cells either were activated with IL-12 without ER- or TSR gene expression was analyzed by RT-PCR. Lane 1, Un- prior incubation in E2 or TS (IL-12) or were not treated with steroids and treated; lane 2, IL-12 activated; lane 3, E2 treated and IL-12 activated; and by guest on September 30, 2021 IL-12 (Control). The production of IFN-␥ from T cells from female and lane 4, TS treated and IL-12 activated. male NOD mice was measured by ELISA (A). The expression of IFN-␥ mRNA by CD4ϩ T cells was analyzed by RT-PCR (B), and the amplified PCR product was normalized against the ␤-actin PCR product (C) (only explanation for this difference in cytokine production between T data from female mice shown). Representative gel pictures (B) or mean Ϯ cells from 4- and 10-wk-old NOD mice is that the T cells might Ͻ ء SD (A and C) of three independent experiments are shown. , p 0.01, express different levels of sex hormone receptors and respond to compared with IL-12-treated control cells. sex hormones according to the numbers of receptors in the cells. Thus, we examined the expression of ER and TSR on CD4ϩ T cells of young (4-wk-old) and adult (10-wk-old) mice. We found found that the most significant difference between sexes was dur- no difference in the level of expression of these receptors at any ing the early stages of insulitis at 4 wk of age; the expression of age, indicating that sex hormone receptors on the CD4ϩ T cells are IFN-␥ was high in females, whereas the expression of IL-4 was not involved in the effect of age on the production of cytokines high in males. A similar result was also reported in a previous from these cells. study (34). Mice approach sexual maturation at 4 wk of age, and Third, we examined whether treatment with estrogen or TS the levels of sex hormones increase in the circulation at this time. would alter the Th1/Th2 (IFN-␥/IL-4) cytokine profile of activated The increased levels of sex hormones at this age probably influ- CD4ϩ T cells from female and male NOD mice. We could not find ence cytokine gene expression. any difference in the production of these cytokines when we acti- Second, we examined the production of Th1/Th2 cytokines in vated T cells with Con A or anti-CD3 Ab in the presence of es- CD4ϩ T cells activated with anti-CD3 Ab in female and male trogen or TS. Therefore, we activated T cells with Con A, rested NOD mice. We found that T cells from 10-wk-old mice produced them in IL-2 for 4 days in the absence of sex hormones, exposed more IFN-␥ and IL-4 than T cells from 4-wk-old mice. This might them to estrogen or TS, and then activated them with IL-12. We be due to the relatively poor activation of T cells from young NOD found that estrogen treatment clearly increased, whereas TS treat- mice as compared with adults (32, 35). However, female mice ment decreased IFN-␥ mRNA expression and IFN-␥ production. produced significantly higher amounts of IFN-␥, but lesser These results suggest that transcriptional regulation of cytokine amounts of IL-4 at 4 wk of age, whereas only a slight difference gene expression is programmed by an initial exposure to sex hor- between females and males was observed at 10 wk of age. There- mones, and reprogram of the gene expression by a different sex fore, the major impact of gender factors on the pathogenesis of hormone requires a long time. An earlier study found that female autoimmune diabetes in NOD mice may be at an early, rather than recipient mice implanted with dihydrotestosterone pellets for later, age. Consistent with this notion, castration at weaning dras- 10Ð14 days before the transfer of encephalitogenic T cells showed tically changed the course of the development of autoimmune di- a significantly less severe course of encephalitis as compared with abetes in both female and male NOD mice (12). One possible control placebo pellet-implanted female mice (36). This may have 5374 ROLE OF SEX HORMONES IN AUTOIMMUNE DIABETES

FIGURE 7. IL-12-induced activation of STAT4 in CD4ϩ T cells from NOD mice treated with E2 or TS. CD4ϩ T cells (5 ϫ 106/ml) were activated by Con A, rested in serum-free medium before treatment with E2 (25 ng/ml) or TS (25 ng/ml) for 20 h, and then activated with IL-12 (20 ng/ml) for 20 min for STAT4 analysis (A and B) or IL-4 (40 ng/ml) for 40 min for STAT6 analysis (C and D). The cell lysate (500 ␮g) was immunoprecipitated with anti-STAT4 Ab (STAT4 IP) or anti-STAT6 (STAT6 IP) Ab, and the immunoprecipitated proteins were further subjected to immunoblot with anti- phosphotyrosine (P-Tyr) Ab. After stripping, the membrane was reprobed with anti-STAT4 or anti-STAT6 Ab. A and C, Lane 1, untreated control; lane Downloaded from 2, IL-12 (A) or IL-4 (C) activated; lane 3, E2 pretreated and IL-12 (A) or IL-4 (C) activated; lane 4, TS pretreated and IL-12 (A) or IL-4 (C) activated. B and D, The phosphorylation of STAT4 (B) or STAT6 (D) was normalized against the amount of STAT4 or STAT6 protein, respectively. Representative gel pictures (A and C) or mean Ϯ SD (B and D) of three independent experiments are shown. Similar results were obtained from CD4ϩ T cells isolated .p Ͻ 0.05, as compared with the IL-12-treated control ,ء .from female and male NOD mice http://www.jimmunol.org/ been due to the enhancement of the Th2 shift by the TS treatment. Acknowledgments In addition, Ag-specific T cells from male donors induced exper- We are grateful to Dr. Merle Olsen for advice and help with animal imental autoimmune encephalomyelitis in female recipients with a care, Lori Bryan for assistance with flow cytometry, and Dr. Ann lower severity than that induced by T cells from female donors L. Kyle for editorial assistance. 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